The present invention relates to liposomes and, more particularly, to liposome vesicles loaded with an active agent.
Liposomes are man-made microscopic vesicles formed from lipid bilayer membranes containing an entrapped aqueous volume. Liposomes may be unilamellar vesicles (possessing a single bilayer membrane) or multilamellar vesicles (onion-like structures characterized by multiple membrane bilayers, each separated from the next by an aqueous layer). The bilayer is composed of two lipid monolayers having a hydrophobic "tail" region and a hydrophilic "head" region. The structure of the membrane bilayer is such that the hydrophobic nonpolar tails of the lipid monolayers orient to the center of the bilayer while the hydrophilic head orients toward the aqueous phase.
The original liposome preparation of Bangham, et al. (J. Med. Biol., 1965, 12:238-252) involves suspending phospholipids in an organic solvent which is then evaporated to dryness leaving a phospholipid film on the reaction vessel. Next, an appropriate amount of an aqueous phase is added, the mixture allowed to swell and the resulting liposomes, which consist of multilamellar vesicles (MLVs) are dispersed by mechanical means. This technique provides the basis for the development of the small sonicated unilamellar vesicles described by Papahadjopoulos et al (Biochem. Biophys. Acta., 1968, 135:624-638), and large unilamellar vesicles
Unilamellar vesicles may be produced using an extrusion apparatus by a method described in Cullis et al., PCT Application No. WO 87/00238, published Jan. 16, 1986, entitled "Extrusion Technique for Producing Unilamellar Vesicles" incorporated herein by reference. Vesicles made by this technique, called LUVETS, are extruded under pressure through a membrane filter.
Another class of liposomes are those characterized as having substantially equal lamellar solute distribution. This class of liposomes is denominated as stable plurilamellar vesicles (SPLV) as defined in U.S. Pat. No. 4,522,803 to Lenk, et al; monophasic vesicles as described in U.S. Pat. No. 4,558,578 to Fountain, et al. and frozen and thawed multilamellar vesicles (FATMLV) wherein the vesicles are exposed to at least one freeze and thaw cycle; this procedure is described in Bally et al., PCT Publication No. 87/00043, Jan. 15, 1987, entitled "Multilamellar Liposomes Having Improved Trapping Efficiencies" and incorporated herein by reference.
A variety of sterols and their water soluble derivatives have been used to form liposomes; see specifically Janoff et al., U.S. Pat. No. 4,721,612 issued Jan. 26, 1988, entitled "Steroidal Liposomes." Mayhew et al., PCT Publication No. WO 85/00968, published Mar. 14, 1985, described a method for reducing the toxicity of drugs by encapsulating them in liposomes comprising alpha-tocopherol and certain derivatives thereof. Also, a variety of tocopherols and their water soluble derivatives have been used to form liposomes, see Janoff et al., PCT Publication No. WO 87/02219, published Apr. 23, 1987, entitled "Alpha Tocopherol-Based Vesicles."
In the present invention, the term lipid as used herein shall mean any suitable material resulting in a bilayer such that a hydrophobic portion of the lipid material orients toward the interior of the bilayer while a hydrophilic portion orients toward the aqueous phase. Lipids further include highly hydrophobic compounds such as triglycerides, sterols such as cholesterol which can be incorporated into the bilayer. The lipids which can be used in the liposome formulations of the present invention are the phospholipids such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidic acid (PA), phosphatidylinositol (PI), sphingomyelin (SPM), and the like, alone or in combination. The phospholipids can be synthetic or derived from natural sources such as egg or soy. Useful synthetic phospholipids are dymyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylglycerol (DMPG). The liposomes can also contain other steroid components such as polyethylene glycol derivatives of cholesterol (PEG-cholesterols), coprostanol, cholestanol, or cholestane, and combinations of PC and cholesterol. They may also contain organic acid derivatives of sterols such as cholesterol hemisuccinate (CHS), and the like. Organic acid derivatives of tocopherols may also be used as liposome-forming ingredients, such as alpha-tocopherol hemisuccinate (THS). Both CHS- and THS-containing liposomes and their tris salt forms may generally be prepared by any method known in the art for preparing liposomes containing these sterols. In particular, see the procedures of Janoff, et al., U.S. Pat. No. 4,721,612 issued Jan. 26, 1988, entitled "Steroidal Liposomes," and Janoff, et al., PCT Publication No. WO 87/02219, published Apr. 23, 1987, entitled "Alpha-Tocopherol Based Vesicles," filed Sept. 24, 1986, respectively. The liposomes may also contain glycolipids.
As indicated above, liposomes can be employed for delivery of a drug. In a liposome drug delivery system, the pharmaceutically active agent is entrapped during liposome formation and then administered to the patient to be treated. The medicament may be soluble in water or in a non-polar solvent.
A liposome drug delivery system is advantageous in that it affords resistance to rapid clearance of the drug accompanied by a sustained release of the drug which will prolong the drug's action. This, in turn, leads to an increased effectiveness of the drug and allows the use of fewer administrations. In the particular case of vaccines, proteins or other immunogens may be entrapped within or in association with liposomes.
In the dried film MLV technique of Bangham et al described above, hereinafter referred to as the Classical Method), lipids are dissolved in a suitable solvent, the solvent rotoevaporated to form a dry lipid film on the flask and the dry film hydrated with an aqueous medium. Lipophilic drugs are incorporated into the liposome by co-dissolving them in the solvent phase while aqueous soluble materials are entrapped from the hydration buffer. While such technique of drug encapsulation is advantageous in that there are not required disruptive applications of heat, sonication, freezing or the addition of solvents (which can facilitate degradation, denaturation, or inactivation of many drugs, especially proteins), a number of disadvantages do exist. In the first place, the resulting product tends to be unstable both in terms of leakage of drug from the capsule into the external aqueous environment and in terms of the presence of oxidation or lyso products. This instability has been attributed to the uneven distribution of drug in the vesicle. More specifically, it has been found that in the onion-like MLVs, the encapsulated drugs tend to be present in high concentrations in the center of the MLV but at low concentrations at the outer layers of the MLV. This concentration differential creates a state of osmotic non-equilibrium and destabilizes the vesicle. Another problem with MLVs prepared by the Classical Method is that only small amounts of drug are sequestered therein, i.e., only between about 5 and 10% of the drug present in the initial solution. This is highly disadvantageous especially when encapsulating very expensive drugs. Yet another problem with the Classical Method is that the formulation of the films along the walls of the reaction vessel renders it difficult to adapt the process to large scale production techniques.
The stable plurilamellar vesicles (SPLVs) described briefly above and in detail in U.S. Pat. No. 4,522,803 represent a significant improvement as compared to the Classical Method in terms of retention of the pharmaceutically active agent and stability. Thus, unlike the MLVs of the classical method, SPLVs are at osmotic equilibrium by virtue of the homogeneous distribution of solute throughout the concentric aqueous spaces of the liposome.
The monophasic vesicles discussed briefly above (MPVs) and described in detail in U.S. Pat. No. 4,588,578 also have an even dispersion of the pharmaceutically active agent throughout the onion-like vesicle structure and, due to the resulting lack of internal osmotic pressure, are relatively stable. Another significant advantage of MPVs is that they can be prepared without resort to sonication or emulsification operations which can adversely affect the active agent. Finally, it has been observed that MPVs may be at least partially resistant to the harsh physical conditions in the gastrointestinal tract thus making this type of vesicle an excellent candidate for applications requiring such resistance.
As is apparent from the above discussion, the art has made significant improvements to liposomes loaded with an active agent in terms of entrapment efficiencies, stability, adaptability to large scale manufacturing techniques, and use of mild conditions to avoid denaturation or other detrimental effects to the pharmaceutically active agent. However, despite such advances, further improvement in the above-listed properties is sought. For example, most of the present techniques for preparing liposomes loaded with an active agent require the active agent to be contacted with the liposome-forming organic solvent. Where the organic solvent adversely affects the active agent, such as by denaturation where the active agent is a protein, the conventional encapsulation techniques are less suitable. Similar adverse effects can occur during repetitive freeze/thaw procedures or other wash procedures.